Acting as an antiplatelet drug, aspirin substantially reduces the occurrence of myocardial infarction and stroke. Despite its remarkable efficacy, some patients do not respond to the drug, raising the question of whether they are resistant its antiplatelet action. This proposal addresses the interindividual variation in the effect of aspirin. Aspirin inhibits prostaglandin H synthase-1 (PGHS-1) by acetylating Ser530 in the catalytic site, thereby irreversibly inhibiting formation of thromboxane A2, an agonist for platelet aggregation. We present novel evidence that acetylation of the PGH synthases by aspirin is highly regulated by the oxidative state of the enzymes;elevation of peroxide concentration antagonizes acetylation, suggesting that it may require hydrogen bonding of aspirin to Tyr385. As Tyr385 is converted to a radical by PGHS peroxidase activity, we propose to examine the participation of PGHS peroxidase activity in the peroxide-induced reversal of acetylation and to characterize the binding of aspirin in the catalytic site with crystallography to ascertain its position in relation to Tyr385. Extending this finding to patients, we will investigate clinical states, including the metabolic syndrome, that are associated with increased hydroperoxide formation and aspirin resistance. The biomarkers that have been associated with aspirin resistance will be evaluated and new metrics for aspirin's effect on its molecular target examined. High levels of excretion of the thromboxane metabolite, 11 -dehydro-thromboxane B2 (TxM), have been associated with an increased risk of coronary events. We now have evidence that a significant portion of TxM in cigarette smokers derives from an extra-platelet source via the inducible PGHS-2 pathway, implying an inflammatory cell origin. The extent to which extra-platelet PGHS-2 derived TxM contributes to high levels of TxM in patients with coronary disease will be determined and correlated with the PGHS-2 promoter polymorphism, with clinical characteristics, and with inflammatory markers. We have found that the initial inhibition of ADP-induced aggregation by aspirin is lost over weeks of treatment. Proposed studies will determine whether this loss of effect of aspirin is dose-dependent, and will further characterize the changes in platelet function associated with the time-dependent loss of effect, including an analysis of changes in platelet protein expression.